Chaperones rescue luciferase folding by separating its domains

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Understanding protein folding is crucial, yet mechanisms for large, multidomain proteins remain largely unknown.
• Large proteins often fold during translation (cotranslational folding) or require molecular chaperones.
• Firefly luciferase serves as a model for cotranslational folding, but its misfolding and chaperone-assisted refolding mechanisms are unclear.

Purpose of the Study:

• To elucidate the refolding mechanism of firefly luciferase.
• To investigate the role of chaperones in preventing and resolving luciferase misfolding.
• To understand how chaperones facilitate the refolding of large, multidomain proteins.

Main Methods:

• Utilized single-molecule force spectroscopy (atomic force microscopy).
• Employed steered molecular dynamics computer simulations.
• Combined experimental and computational approaches to study luciferase refolding.

Main Results:

• Partially unfolded luciferase can refold without chaperones if the N-terminal domain remains intact.
• Complete unfolding leads to stable, non-native configurations due to inter-domain interactions.
• Chaperones enable rapid refolding of completely unfolded luciferase by sequestering non-native residues.

Conclusions:

• Chaperones prevent misfolding by sequestering exposed hydrophobic residues, facilitating sequential domain folding.
• Chaperone action may mimic the ribosomal exit tunnel, promoting cotranslational-like folding pathways.
• This mechanism provides insights into the folding of large proteins in vivo.